A detailed mapping of calretinin positive cells and fibers in the rat thalamus was completed using in situ hybridization histochemistry and immunocytochemistry. Results of this study revealed populations of calretinin positive cells in several discrete thalamic nuclei (e.g., reticular, rhomboid, reuniens, paraventricular) and in regions which overlapped defined nuclear thalamic boundaries (e.g., cells in the central and intralaminar nuclei continuous with the central grey). Unilateral cochlea ablations were found to increase the immunoreactivity of neurons in the ipsilateral ventral cochlear nucleus and contralateral trapezoid body. However, no changes were seen in mRNA label. In situ hybridization histochemistry revealed that calretinin mRNA is present early in development (E9) in chick embryos. A 39 kDa protein, whose phosphorylation is inhibited by calretinin, was found predominantly in mitochondrial membranes and was localized in several peripheral tissues with greatest amounts in the testis. Calretinin inhibited the phosphorylation of this protein in all regions and subcellular fractions where the 39 kDa band was visible on autoradiograms. Divalent cations stimulated both the phosphorylation of the 39 kDa (Mg2+, Mn2+ , Ni 2+, Ca2+ or Co2+) and the inhibition by calretinin (Mg2+, Co2+ or Ni 2+). Zn2+ inhibited both the phosphorylation of the 39 kDa band and the effect on calretinin, while EDTA attenuated the calretinin effect. Calretinin also produced a slight attenuation in the phosphorylation of a 44 kDa band identified as the alpha subunit of pyruvate dehydrogenase. In contrast, initial examination of the effects of a related calcium binding protein (calbindin D-28k) revealed a stimulation of protein phosphorylation and a reversal of the inhibitory effect of calretinin on phosphorylation of the 39 kDa protein.